Efficient Labeling of Estrogen Receptor α Inside Cells using Affimer, an Antibody Mimetic

Estrogen receptor α (ERα) is expressed in around 80% of breast cancers and is associated with proliferation and tumor growth. Mutations in ERα, like Y537S, are associated with driving metastasis and poor disease-free survival. However, the interaction of ERα with different ligands remain to be fully understood. Here we use a small antibody mimetic, a 13 kDa anti-ERα affimer conjugated to an organic fluorophore (Alexa647), to label a cell containing the wild-type ERα (ERαWT), the mutant ERαY537S, or the ERαL536S, a non-activatable mutant. Intracellular labeling was done by treating the cell with Streptolysin O to create temporary pores in the membrane, allowing the cell to be healthy after repairing the pores. We demonstrate that the affimer labelling efficiency for ERαWT increases from 50% to 80% after estradiol (E2) treatment; for ERαY537S staining almost complete (80-90%) with or without E2; and for ERαL536S, very little staining is observed. These findings indicate the affimer has a higher affinity towards the “active” conformations of ERα. We also tested affimer labeling of ERαWT when using three ligands: two selective ER modulators (SERMs), 4-hydrotamoxifen (4-OH) and 27-Hydroxycholesterol (27HC), and a selective ER degrader (SERD), ICI. Affimer-labeling for ERαWT decreases in the presence of 4-OH and ICI treatment, but increases with 27HC treatment. As a control, the effects of these ligands are tested via a luciferase assay utilizing the estrogen responsive elements (ERE). Both results show that for ERαWT, 4-OH and ICI can inhibit the agonistic function of E2, while 27HC has similar estrogenic activity to E2. Similar measurements are presented for the mutants. In summary, we demonstrate the affimer is a reliable small antibody mimetic which can efficiently label active ERαWT and ERαY537S in the presence of different ligands in mammalian cells.